In vitro assessment of cetalkonium chloride induced damage in human corneal keratocytes

This study aimed to conduct an in vitro assessment of the concentration- and time-dependent cytotoxic effects of cetalkonium chloride (CKC), a preservative used in ophthalmic preparations, on human corneal keratocytes and to investigate its underlying cellular mechanisms of toxicity. Primary human corneal keratocytes were treated with a range of CKC concentrations (0 to 4.0 × 10^-4% w/v) for up to 72 h, after which cell viability, intracellular reactive oxygen species (ROS) production, mitochondrial integrity, and the expression of key proteins in cell survival and apoptosis pathways were analyzed. The results showed that CKC induced a significant, concentration- and time-dependent reduction in keratocyte viability and a dose-dependent increase in ROS production. CKC exposure also led to the collapse of the mitochondrial network, cell shrinkage. Mechanistically, CKC suppressed the phosphorylation of pro-survival proteins Akt, mTOR, and ERK, while increasing the pro-apoptotic BAX/Bcl-xL ratio. In conclusion, CKC exhibits toxicity to keratocytes through mechanisms involving increased ROS, mitochondrial damage, cell membrane damage, and alterations of key survival/apoptosis signaling pathways. These findings provide a critical basis for defining safe concentration limits for CKC, guiding the development of ophthalmic preservative systems that minimize stromal keratocyte damage.